Methods of generating hyper inos expressing cells and uses thereof

ABSTRACT

A method of generating a hyper iNOS expressing cell includes administering to a myeloid derived cell an amount of a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonist effective to substantially inhibit STAT3 activation in the cell and administering an inflammatory insult to the cell to stimulate hyper iNOS expression from the cell.

RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.13/780,503, filed Feb. 28, 2013, which is a Continuation of U.S.application Ser. No. 13/578,785, filed Aug. 13, 2012, which is aNational Phase filing of PCT/US2011/024757, filed Feb. 14, 2011, andclaims priority from U.S. Provisional Application No. 61/303,871, filedFeb. 12, 2010, 61/320,879 filed Apr. 5, 2010, 61/434,151, filed Jan. 19,2011, the subject matter of which are incorporated herein by referencein their entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. RR024990awarded by The National Institute of Health. The United Statesgovernment has certain rights to the invention.

TECHNICAL FIELD

This application relates to the generation of hyper iNOS (HiNOS)expressing myeloid cells and their use in therapeutic methods, methodsof inhibiting wound healing, and an animal model of delayed woundhealing.

BACKGROUND

Monocytes/macrophages demonstrate topographical and functionalspecificity directed by the micro-anatomic milieu suggesting a key roleof extracellular factors in determining cell differentiation andfunctional activity. Interactions with the microenvironment results inmacrophage activation, production of numerous soluble signalingmolecules including pro- and anti-inflammatory cytokines, and growth andregulatory factors. Following injury, macrophages and monocytes arerecruited and become key mediators of inflammation, tissue repair, andcellular debris clearance. However, dysfunctional control of themagnitude and duration of inflammation can result in damage to the host,such as in auto-immunity, numerous destructive and degenerative diseases(rheumatoid arthritis and Alzheimer's disease), non-healing ulcers, andinfections such as leprosy and leishmaniasis. A hallmark of excessinflammation is damage to the surrounding tissue. However, the factorsand underlying mechanisms that govern the function of macrophages in thecontext of inflammation and tissue destruction remain incompletelyunderstood.

SUMMARY

An aspect of the application relates to a method of generating a hyperiNOS expressing cell. The method includes administering to a myeloidderived cell an amount of a PPARγ agonist and an IL-6/STAT3 signalingpathway antagonist effective to substantially inhibit STAT3 activationin the cell, and administering an inflammatory insult to the cell tostimulate hyper iNOS expression from the cell.

Another aspect of the application relates to a method of mediating localdestruction of tissue in a subject. The method includes administering anamount of a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonistto macrophages of the subject effective to substantially inhibit STAT3activation in the macrophages of the subject; and administering anamount of an inflammatory or damaging insult to the tissue of thesubject effective to induce hyper iNOS expression of the macrophages inor about the periphery of the tissue.

A further aspect of the application relates to a method of treatingcancer or a tumor in a subject. The method includes administering anamount of a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonistto the subject effective to substantially inhibit STAT3 activation inmacrophages of the subject; and administering an amount of aninflammatory or damaging insult to the cancer or tumor of the subjecteffective to induce hyper iNOS expression of the macrophages in or aboutthe periphery of the cancer or tumor.

A still further aspect of the application relates to a method oftreating an infection in a subject. The method includes administering anamount of a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonistto macrophages of the subject effective to substantially increase iNOSexpression of macrophages at the site of infection.

Yet another aspect of the application relates to a method for inhibitingunwanted wound repair in a subject. The method includes administering anamount of a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonistto the subject effective to substantially increase iNOS expression ofmacrophages at the subject's wound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematic representations of mechanisms modulatinginflammatory responses in (A) normal macrophages and (B), in the absenceof IL-6, in hyper-inflammatory macrophages.

FIG. 2 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression of bone marrow derived macrophages (BMDMs) isolated fromC57BL/6 and IL6^(−/−) mice stimulated with PPARγ agonist and/or LPS.

FIG. 3 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression of in vivo elicited peritoneal macrophages isolated fromC57BL/6 and IL6^(−/−) mice stimulated with PPARγ agonist and/or LPS.

FIG. 4 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression of in vivo elicited peritoneal macrophages treated witheither rosiglitazone (Rosi) or Pioglitazone (Pio).

FIG. 5 illustrates a graph showing iNOS expression of in vivo elicitedperitoneal macrophages treated with either rosiglitazone (Rosi) or aninhibitor of PPARγ signaling, GW9662.

FIG. 6 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression of mice subjected to intratracheal bleomycin or treated withPPARγ agonist.

FIG. 7 illustrates (A) a graph showing the ratio of NFκBp65 to STAT3after LPS stimulation with and without PPARγ agonist treatment and (B) aWestern blot of NFκBp65 and STAT3 proteins extracted at 30 mins and 60mins from WT and IL6^(−/−) peritoneal macrophages.

FIG. 8 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression from lungs of C57BL/6 and IL6^(−/−) mice subjected tointratracheal bleomycin or treated with PPARγ agonist.

FIG. 9 illustrates confocal images of immunostaining for colocalizationof iNOS with macrophage markers CD11b⁺ & F4/80⁺ in (A) wildtype and (B)IL6^(−/−) mice lung sections.

FIG. 10 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression from lungs of C57BL/6 and IL6^(−/−) mice subjected to acutaneous wounding protocol and treated with PPARγ agonist.

FIG. 11 illustrates confocal images of immunostaining for colocalizationof iNOS with CD11b⁺ & F4/80⁺ cells in (A) C57BL/6 and (B) IL6^(−/−) skinsections.

FIG. 12 illustrates images showing H&E staining of wildtype (A) C57Bl/6and (B) IL6^(−/−) mice skin tissue surrounding the wound margin at 0, 9,and 24 hours following wounding; (C) confocal images of immunostainingfor F4/80 and CD11b double positive cells in C57Bl/6, and (D), IL6^(−/−)mice skin sections.

FIG. 13 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression during time course of wildtype C57Bl/6 and IL6^(−/−) miceskin tissue.

FIG. 14 illustrates images showing (A) iNOS staining colocalized withF4/80⁺ cells, but not with LY6/GR1⁺ cells, and H&E staining of skinafter (B) PBS- or (C) clodronate-filled liposomes were injectedintradermally surrounding the wound edge in IL6^(−/−) mice.

FIG. 15 illustrates (A) images showing wounds of mice subjected to awounding protocol including Application of a PPARγ agonist on IL6^(−/−)animals; (B) a wound healing graph showing the mean percent of initialwound; (C) images showing H&E staining of wounded skin with and withoutanti-CD11b or isotype control i.p. injection; and (D) confocal images ofimmunostaining for F4/80, CD11b, and nucleus. Arrows indicate doublepositive cells.

FIG. 16 illustrates graphs showing (A) iNOS expression and (B) TNF-γexpression of skin surrounding the wound edge of IL6^(−/−) mice.

FIG. 17 illustrates wound healing graphs showing restored wound healingcapacity of IL6^(−/−) mice under experimental conditions when (A)treated with anti-CD11b to prevent monocyte/macrophage infiltration, (B)treated with the iNOS specific inhibitor, 1400 W, or (C) treated with ananti-TNF-γ antibody administered by i.p. injection 1 hour beforewounding.

FIG. 18 illustrates images showing (A-C) WT and (D-F) IL-6^(−/−) micelungs harvested and stained for CD11b, F4/80 markers of macrophages, andiNOS.

FIG. 19 illustrates graphs showing PPARγ agonist treatment of IL-6^(−/−)mice delays wound healing and increases iNOS expression in skin. Woundsof IL-6^(−/−) mice expand above 100% after treatment and remain abovebaseline after treatment with PPARγ agonist and UVB compared to thewounds of wildtype or IL-6^(−/−) mice treated with (A) PPARγ agonistonly; (B) UVB and vehicle; (C) vehicle only; and (D) wound only.

FIG. 20 illustrates a chart showing iNOS expression of ex vivo elicitedperitoneal macrophages from WT (C57Bl/6 mice) plated in 24 well plates,treated with Rosiglitazone (5 uM) and/or Simvistatin (Sim) or Mevistatin(Mev) at 0.1, 1, and 10 μM concentrations for 16 hours, and thenstimulated with 1 ng/mL LPS for 4 hours.

FIG. 21 illustrates a wound healing graph showing WT (c57BL/6) mice aged6-8 weeks subjected to a cutaneous wounding protocol and injected with0.6 mg of simvistatin 2 hours prior to wounding and treated topicallywith a 1% solution of rosiglitazone in 10 g Aquaphor. Wounds weremeasured and treated with topical rosiglitazone solution daily untilhealed (loss of serum crust and reepithelialization).

FIG. 22 illustrates a graph showing iNOS expression in human macrophagestreated with statin, anti-CD11b, and PPAR.

FIG. 23 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression of peritoneal macrophages isolated from C57BL/6 and IL6^(−/−)mice treated in vivo with a PPARγ agonist as indicated.

FIG. 24 illustrates images of (A, B) B16 melanoma cells seeded for 24hours prior to introduction of either WT or IL-6^(−/−) macrophages at a4:1 E:T ratio of 2 million macrophages (pre-condition with PPARγagonists and stimulated with LPS) to 500,000 melanoma cells, (C) 48hours after co-seeding melanoma with the WT macrophages, and (D) 48hours after co-seeding the melanoma with IL-6^(−/−) macrophages revealeda drastic reduction of tumor cells.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent application pertains. Commonly understood definitions ofmolecular biology terms can be found in, for example, Lodish et al.,Molecular Cell Biology, 6th Edition, W. H. Freeman: New York, 2007, andLewin, Genes IX, Jones and Bartlett Publishers: Mass., 2008. Forconvenience, certain terms employed in the specification, examples, andappended claims are collected here.

As used herein, “one or more of a, b, and c” means a, b, c, ab, ac, bc,or abc. The use of “or” herein is the inclusive or.

As used herein, “protein” is a polymer consisting of the 20 amino acids.Although “polypeptide” is often used in reference to relatively largepolypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and is varied.

The terms “polynucleotide sequence” and “nucleotide sequence” are alsoused interchangeably herein.

“Recombinant,” as used herein, means that a protein is derived from aprokaryotic or eukaryotic expression system.

The term “wild type” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo.

As used herein, the term “nucleic acid” refers to polynucleotides, suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide,including both exon and (optionally) intron sequences.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. Preferred vectors are those capable of one or more of,autonomous replication and expression of nucleic acids to which they arelinked. Vectors capable of directing the expression of genes to whichthey are operatively linked are referred to herein as “expressionvectors”.

A polynucleotide sequence (DNA, RNA) is “operatively linked” to anexpression control sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed, and maintaining the correct reading frame topermit expression of the polynucleotide sequence under the control ofthe expression control sequence, and production of the desiredpolypeptide encoded by the polynucleotide sequence.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to nucleic acid sequences, such as initiationsignals, enhancers, and promoters, which induce or control transcriptionof protein coding sequences with which they are operably linked. In someexamples, transcription of a recombinant gene is under the control of apromoter sequence (or other transcriptional regulatory sequence) whichcontrols the expression of the recombinant gene in a cell-type in whichexpression is intended. It will also be understood that the recombinantgene can be under the control of transcriptional regulatory sequenceswhich are the same or which are different from those sequences, whichcontrol transcription of the naturally occurring form of a protein.

As used herein, the term “tissue-specific promoter” means a nucleic acidsequence that serves as a promoter, i.e., regulates expression of aselected nucleic acid sequence operably linked to the promoter, andwhich affects expression of the selected nucleic acid sequence inspecific cells of a tissue, such as cells of epithelial cells. The termalso covers so-called “leaky” promoters, which regulate expression of aselected nucleic acid primarily in one tissue, but cause expression inother tissues as well.

“Homology” and “identity” are used synonymously throughout and refer tosequence similarity between two peptides or between two nucleic acidmolecules. Homology can be determined by comparing a position in eachsequence, which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are homologous or identical at that position. Adegree of homology or identity between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a polypeptide with a second amino acid sequencedefining a domain (e.g., polypeptide portion) foreign to and notsubstantially homologous with the domain of the first polypeptide. Achimeric protein may present a foreign domain which is found (albeit ina different protein) in an organism which also expresses the firstprotein, or it may be an “interspecies”, “intergenic”, etc. fusion ofprotein structures expressed by different kinds of organisms.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, which are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material orculture medium when produced by recombinant DNA techniques, or chemicalprecursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments,which are not naturally occurring as fragments and would not be found inthe natural state.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intraventricular, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into a target tissue (e.g., the central nervoussystem), such that it enters the animal's system and, thus, is subjectto metabolism and other like processes, for example, subcutaneousadministration.

As used herein, the term “agent” or “drug” is used herein to denote achemical compound, a mixture of chemical compounds, a biologicalmacromolecule, or an extract made from biological materials such asbacteria, plants, fungi, or animal particularly mammalian) cells ortissues that are suspected of having therapeutic properties. The agentor drug may be purified, substantially purified or partially purified.As used herein, the term “purified” or “to purify” refers to the removalof one or more contaminants from a sample. As used herein, the term“partially purified” refers to the removal of a moderate portion of thecontaminants of a sample to the extent that the substance of interest isrecognizable by techniques known to those skilled in the art asaccounting for a measurable amount of the mixture. Preferably, thecompound of interest is at least 5% of the total preparation and up to50% of the total preparation. As used herein, the term “substantiallypurified” refers to the removal of a significant portion of thecontaminants of a sample to the extent that the substance of interest isrecognizable by techniques known to those skilled in the art as the mostabundant substance in the mixture.

As used herein, the term “therapeutically effective amount” refers tothat amount of a composition, which results in amelioration of symptomsor a prolongation of survival in a patient. A therapeutically relevanteffect relieves to some extent one or more symptoms of a disease orcondition or returns to normal either partially or completely one ormore physiological or biochemical parameters associated with orcausative of the disease or condition.

As used herein “agonist” refers to molecules or compounds which mimicthe action of a “native” or “natural” compound. Agonists may behomologous to these natural compounds in respect to conformation, chargeor other characteristics. Thus, agonists may be recognized by, e.g.,nuclear receptors. This recognition may result in physiologic and/orbiochemical changes within the cell, such that the cell reacts to thepresence of the agonist in the same manner as if the natural compoundwas present.

As used herein “antagonist” refers to molecules or compounds whichdecrease or inhibits the expression and/or function of a “native” or“natural” compound. An antagonist may be a type of receptor ligand ordrug (e.g., a receptor antagonist) that does not provoke a biologicalresponse itself upon binding to a receptor at an active site or toallosteric sites on receptors, or they may interact at unique bindingsites not normally involved in the biological regulation of thereceptor's activity, but blocks or dampens agonist-mediated responses.

As used herein, the term “PPARγ agonist” refers to a compound orcomposition, which when combined with peroxisome proliferator-activatedreceptor gamma (PPARγ), directly or indirectly stimulates or increasesan in vivo or in vitro reaction typical for the receptor (e.g.,transcriptional regulation activity). The increased reaction can bemeasured by any of a variety of assays known to those skilled in theart. An example of a PPARγ agonist is a thiazolidinedione compound, suchas troglitazone, rosiglitazone, pioglitazone, ciglitazone, WAY-120,744,englitazone, AD 5075, darglitazone, and congeners, analogs, derivatives,and pharmaceutically acceptable salts thereof.

As used herein, the term “IL-6/STAT3 signal pathway antagonist” refersto any agent that substantially decreases or inhibits the expressionand/or function of a component of the IL-6/STAT3 signaling pathway in amyeloid cell.

As used herein, the term “IL-6 antagonist” refers to any agent thatsubstantially decreases the expression and/or function of IL-6 in asubject. An example of an IL-6 antagonist is a statin.

As used herein, the term “subject” refers to any mammal, such as humanbeings, rats, mice, cats, dogs, goats, sheep, horses, monkeys, apes,rabbits, cattle, etc. The mammalian subject can be in any stage ofdevelopment including adults, young animals, and neonates. Mammaliansubjects can also include those in a fetal stage of development.Typically, the terms “patient” and “subject” are used interchangeablyherein in reference to a human subject.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments consist of, but are not limited to,test tubes and cell culture. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionthat occur within a natural environment.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not substantially develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms. Treatment, prevention and ameliorating acondition, as used herein, can include, for example decreasing oreradicating a deleterious or harmful condition associated with unwantedwound repair, unwanted tissue, pathogenic infection, neoplasia,inflammation, and delayed wound healing.

As used herein, the term “promoting wound healing” or “promoting healingof a wound” mean augmenting, improving, increasing, or inducing closure,healing, restoring normal wound healing or repair of a wound.

This application relates to immunotherapeutic methods of promoting arobust innate immune response in a subject. This application alsorelates to the use of a subject's innate immunity to combat infection,unwanted tissue, and neoplastic disease through the selective generationof highly inflammatory hyper iNOS expressing myeloid cells (HiNOS). TheHiNOS cells in accordance with the application can be generated throughcombinatory stimulation of a myeloid cell's PPARγ receptors andinhibition of the IL-6/STAT3 signaling pathway in the presence of aninflammatory or damaging insult or signal.

It was found that treatment of myeloid cells (or myeloid derived cells)with a PPARγ agonist in combination with inhibition of the IL-6/STAT3signaling pathway in vitro and in vivo results in synergistichyper-inflammatory responses, generation of pro-inflammatory, hyper-iNOSand TNF-γ expressing macrophages, and drastic macrophage-mediated denovo tissue destruction. The net response of PPARγ ligand exposure andconcomitant IL-6 deficiency primes the myeloid cells so that during aperiod of stimulation, such as an inflammatory or damaging insult, theprimed cells hyper express iNOS.

As illustrated schematically in FIG. 1A, monocytes and macrophagesinfiltrating inflamed tissue are exposed to the proinflammatory cytokineTNF-α which in turn activates NF-κB signaling resulting in induction ofiNOS. To limit excessive exposure to TNF-α, cells release soluble TNFreceptor, shielding them from excess stimulation and further NF-κBactivation, a response that is diminished in cells deficient in IL-6.The addition of a PPARγ ligand, such as a thiazolidinedione, altersanother aspect of NF-κB activity, specifically STAT3-mediatedtransrepression of NF-κB. As illustrated schematically in FIG. 1B,inhibition of STAT3 activation decreases its nuclear accumulation and inturn reduces its ability to control the magnitude of NF-κB responsivegenes, such as iNOS. We found that excessive inflammation propagated bythese HiNOS macrophages resulting in tissue destruction and ultimatelyleads to necrosis and delayed tissue repair as often seen in humanchronic wounds. The HiNOS macrophages can be induced in several modelsystems suggesting that these findings are clinically relevant to alarge number of patients, given the common concurrence of relativestates of IL-6 inhibition with PPARγ activation induced either bypharmacologic agonists or by endogenous ligands generated duringinflammation and oxidant stress.

One aspect of the application, therefore relates to a method ofgenerating HiNOS cells. The method includes administering to a myeloidderived cell an amount of a PPARγ agonist and an IL-6/STAT3 signalingpathway antagonist effective to substantially inhibit STAT3 activationin the myeloid cell in combination with administering an inflammatoryinsult or signal to the myeloid cell to stimulate hyper iNOS expressionfrom the cell. By substantially inhibiting STAT3 activation, it is meantthe STAT3 activation in the myeloid derived cell treated with the PPARγagonist and the IL-6/STAT3 signaling pathway antagonist is inhibited atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% compared tountreated myeloid cells under similar in vivo or in vitro environmentalor physiological conditions. The myeloid derived cell can be, forexample, a macrophage or monocyte and the iNOS expression can beincreased at least 2 fold, at least 5 fold, at least 10 fold, at least20 fold, at least 30 fold, at least 40 fold, or at least 50 foldcompared an untreated myeloid derived cell.

In some aspects, HiNOS cells can be generated in vitro. Myeloid cellsfor generating HiNOS cells in vitro can be obtained from bone marrow,peritoneal, bronchial lavage, and hepatic sources. In one non-limitingexample, bone marrow derived macrophages can be harvested from asubject's femur using well known methods. Macrophages can then beisolated and cultured in vitro with the addition of macrophage-colonystimulating factor (MCSF). The cells can then stimulated with aninflammatory insult, such as by administering LPS, anti-CD11b antibody,or complement proteins, in combination with administration of a PPARγagonist and an IL-6/STAT3 signaling pathway antagonist.

In some aspects, in vitro generated HiNOS cells derived from a subjectcan be administered back to the subject in a therapeutic method. Forexample, in vitro generated HiNOS cells can be administered systemicallyor implanted in a region of interest depending on the therapeuticpurpose.

In other aspects of the application, HiNOS cells can be generated invivo. For example, a subject can be administered a PPARγ agonist and anIL-6/STAT3 signaling pathway antagonist to substantially inhibit STAT3activation in myeloid cells (e.g., macrophages) of the subject and themacrophages treated with the PPARγ agonist and an IL-6/STAT3 signalingpathway antagonist can be stimulated with an inflammatory or damaginginsult to induce hyper iNOS expression from the cells.

In certain embodiments, the IL-6/STAT3 signaling pathway antagonist caninclude any agent or composition that substantially decreases orinhibits the expression and/or functional activity of a component of theIL-6/STAT3 signaling pathway in a myeloid cell. The functional activityof the IL-6/STAT3 signaling pathway can be suppressed, inhibited, and/orblocked in several ways including: direct inhibition of the activity ofIL-6 and/or STAT3 (e.g., by using neutralizing antibodies, smallmolecules or peptidomimetics, dominant negative polypeptides);inhibition of genes that express IL-6 and/or STAT-3 (e.g., by blockingthe expression or activity of the genes and/or proteins); activation ofgenes and/or proteins that inhibit one or more of the functionalactivity of IL-6 and/or STAT3 (e.g., by increasing the expression oractivity of the genes and/or proteins); inhibition of genes and/orproteins that are downstream mediators of the iNOS expression (e.g., byblocking the expression and/or activity of the mediator genes and/orproteins); introduction of genes and/or proteins that negativelyregulate one or more of functional activity of IL-6 and/or STAT3 (e.g.,by using recombinant gene expression vectors, recombinant viral vectorsor recombinant polypeptides); or gene replacement with, for instance, ahypomorphic mutant of STAT-3 (e.g., by homologous recombination,overexpression using recombinant gene expression or viral vectors, ormutagenesis).

In an embodiment of the application, the IL-6/STAT3 signaling pathwayantagonist is an IL-6 antagonist. In some aspects, the IL-6 antagonistcan include a humanized IL-6 receptor-inhibiting monoclonal antibody. Incertain aspects, the IL-6 antagonist is the product tocilizumab (adescriptive name sold under the trademark ACTEMRA® by Roche,Switzerland). In other aspects, the IL-6 antagonist can include avaccine that when administered to a subject generates IL-6 antibodies inthe subject. An example of such a vaccine is disclosed in Fosergau etal. Journal of Endocrinology (2010) 204, 265-273.

In another embodiment, the IL-6/STAT3 signaling pathway antagonist is atyrosine kinase inhibitor. Exemplary tyrosine kinase inhibitors for usein the present invention include but are not limited to tyrphostins, inparticular AG-490, and inhibitors of Jak, Src, and BCR-Abl tyrosinekinases. Other tyrphostins suitable for use herein include, but are notlimited to AG17, AG213 (RGS0864), AG18, AG82, AG494, AG825, AG879,AG1112, AG1296, AG1478, AG126, RG13022, RG14620, AG555, and relatedcompounds. In certain aspects, a BCR-Abl tyrosine kinase inhibitor foruse herein can include the product imatinib mesilate (a descriptive namesold under the trademark GLEEVEC® by Novartis, Switzerland).

In a further embodiment, the IL-6/STAT3 signaling pathway antagonist isan HMG CoA reductase inhibitor (3-hydroxymethylglutaryl coenzyme Areductase inhibitors) (e.g., statin). HMG-CoA (3-hydroxy methylglutarylcoenzyme A) reductase is the microsomal enzyme that catalyzes the ratelimiting reaction in cholesterol biosynthesis (HMG-CoA Mevalonate).Statins can inhibit and/or reduce IL-6 expression when administered tomyeloid cells (e.g., macrophages) and as shown in Examples 3 and 4 canbe used in combination with PPARγ agonists to generate hyper-INOSexpressing macrophages.

Statins that can be used for administration, or co-administration withother agents according to the invention include, but are not limited to,simvastatin (U.S. Pat. No. 4,444,784), mevistatin, lovastatin (U.S. Pat.No. 4,231,938), pravastatin sodium (U.S. Pat. No. 4,346,227),fluvastatin (U.S. Pat. No. 4,739,073), atorvastatin (U.S. Pat. No.5,273,995), cerivastatin, and numerous others described in U.S. Pat. No.5,622,985, U.S. Pat. No. 5,135,935, U.S. Pat. No. 5,356,896, U.S. Pat.No. 4,920,109, U.S. Pat. No. 5,286,895, U.S. Pat. No. 5,262,435, U.S.Pat. No. 5,260,332, U.S. Pat. No. 5,317,031, U.S. Pat. No. 5,283,256,U.S. Pat. No. 5,256,689, U.S. Pat. No. 5,182,298, U.S. Pat. No.5,369,125, U.S. Pat. No. 5,302,604, U.S. Pat. No. 5,166,171, U.S. Pat.No. 5,202,327, U.S. Pat. No. 5,276,021, U.S. Pat. No. 5,196,440, U.S.Pat. No. 5,091,386, U.S. Pat. No. 5,091,378, U.S. Pat. No. 4,904,646,U.S. Pat. No. 5,385,932, U.S. Pat. No. 5,250,435, U.S. Pat. No.5,132,312, U.S. Pat. No. 5,130,306, U.S. Pat. No. 5,116,870, U.S. Pat.No. 5,112,857, U.S. Pat. No. 5,102,911, U.S. Pat. No. 5,098,931, U.S.Pat. No. 5,081,136, U.S. Pat. No. 5,025,000, U.S. Pat. No. 5,021,453,U.S. Pat. No. 5,017,716, U.S. Pat. No. 5,001,144, U.S. Pat. No.5,001,128, U.S. Pat. No. 4,997,837, U.S. Pat. No. 4,996,234, U.S. Pat.No. 4,994,494, U.S. Pat. No. 4,992,429, U.S. Pat. No. 4,970,231, U.S.Pat. No. 4,968,693, U.S. Pat. No. 4,963,538, U.S. Pat. No. 4,957,940,U.S. Pat. No. 4,950,675, U.S. Pat. No. 4,946,864, U.S. Pat. No.4,946,860 U.S. Pat. No. 4,940,800, U.S. Pat. No. 4,940,727, U.S. Pat.No. 4,939,143, U.S. Pat. No. 4,929,620, U.S. Pat. No. 4,923,861, U.S.Pat. No. 4,906,657, U.S. Pat. No. 4,906,624 and U.S. Pat. No. 4,897,402,the disclosures of which patents are incorporated herein by reference.

In yet another embodiment, the IL-6/STAT3 signaling pathway antagonistcan be a STAT3 inhibitor. Examples of STAT3 inhibitors are described inU.S. Patent Application No. 2010/0041685 and can include4-[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-3-oxo-1-propen-1-yl]benzoicacid;4{5-[(3-ethyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-fu-ryl}benzoicacid;4-[({3-[(carboxymethyl)thio]-4-hydroxy-1-naphthyl}amino)sulfonyl]benzoicacid;3-({2-chloro-4-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-6-ethoxyphenoxy}methyl)benzoicacid; methyl4-({[3-(2-methyoxy-2-oxoethyl)-4,8-dimethyl-2-oxo-2H-chromen-7-yl]oxy}met-hyl)benzoate;4-chloro-3-{5-[(1,3-diethyl-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidiny-lidene)methyl]-2-furyl}benzoicacid; a functionally active derivative thereof and a mixture thereof.Other examples of STATS inhibitors are described in WO 2010/118309 andin G. Zinzalla et al. Bioorg. Med. Chem. Lett. 20 (2010)7029-7032.

In certain embodiments, the PPARγ agonist can include any agent orcomposition that substantially increases or promotes the activationand/or functional activity of PPARγ. The functional activity of thePPARγ can be increased or promoted in several ways including: directactivation of PPARγ with an endogenous PPARγ ligand (e.g., by usingsmall molecules or peptidomimetics); up regulation of genes for ligandsof PPARγ (e.g., by promoting the expression or activity of the genesand/or proteins); activation of genes and/or proteins that promote oneor more of the functional activity of PPARγ (e.g., by increasing theexpression or activity of the genes and/or proteins); promotion of genesand/or proteins that are downstream mediators of PPARγ activation; andintroduction of genes and/or proteins that promote one or more offunctional activity of PPARγ (e.g., by using recombinant gene expressionvectors, recombinant viral vectors or recombinant polypeptides).

In other embodiments, PPARγ agonists that can be used herein include,for example, prostaglandin J2 (PGJ2) and analogs thereof (e.g.,A2-prostaglandin J2 and 15-deoxy-2,4-prostaglandin J2), members of theprostaglandin D2 family of compounds, docosahexaenoic acid (DHA), andthiazolidinediones (e.g., ciglitazone, troglitazone, pioglitazone androsiglitazone).

In some embodiments, the PPARγ agonist can include a thiazolidinedioneor a derivative thereof. In some aspects, a PPARγ agonist for use in thepresent invention includes at least one compound or a pharmaceuticallysalt thereof selected from the group consisting of:(+)-5[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4thiazolidinedione;5-[4-[2-(5-ethylpyridin-2-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;(ciglitazone); 4-(2-naphthylmethyl)-1,2,3,5-oxathiadiazole-2-oxide;5-[4-[2-[(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]-5-methlthiazolidine-2,4-dione;5-[4-[2-[2,4dioxo-5-phenylthiazolidine-3-yl)ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-[(N-methyl-N-(phenoxycarbonyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-phenoxyethoxy)benzyl]thiazolidine-2,4-dione;5-[4-[2-(4-chorophenyl)ethylsulfonyl]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[[4-(3-hydroxy-1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl]thiazolidine-2,4-dione;5-[[2-(2-naphthylmethyl)benzoxazo]-5-ylmethyl]thiazolidine-2,4-dione;5-[4-[2-(3-phenylureido)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[2-(N-benzoxazol-2-yl)-N-metholamino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[2-(5-methyl-2-phenyloxazol-4-ylmethyl)benzofuran-5-ylmethyl]oxazolidine-2,4-dione;5-[4-[2-(N-methyl-N-(2-pyridyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;and5-[4-[2-(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]oxazolidine-2,4-dione.In certain aspects, the PPARγ agonist is the product rosiglitazone (adescriptive name sold under the trademark AVANDIA® by GlaxoSmithKline,U.K.).

In other embodiments, PPARγ agonists can include, but are not limitedto, L-tyrosine-based compounds, farglitazar, GW7845, indole-derivedcompounds, indole 5-carboxylic acid derivatives and 2,3-disubstitutedindole 5-phenylacetic acid derivatives. It is significant that most ofthe PPARγ agonists exhibit substantial bioavailability following oral ortopical administration and have little or no toxicity associated withtheir use (See, e.g., Saltiel and Olefsky, Diabetes 45:1661 (1996); Wanget al., Br. J. Pharmacol. 122:1405 (1997); and Oakes et al., Metabolism46:935 (1997)).

PPARγ agonists that can be used for practicing the present invention,and methods of making these compounds, are disclosed in WO 91/07107; WO92/02520; WO 94/01433; WO 89/08651; WO 96/33724; WO 97/31907; U.S. Pat.Nos. 4,287,200; 4,340,605; 4,438,141; 4,444,779; 4,461,902; 4,572,912;4,687,777; 4,703,052; 4,725,610; 4,873,255; 4,897,393; 4,897,405;4,918,091; 4,948,900; 5,002,953; 5,061,717; 5,120,754; 5,132,317;5,194,443; 5,223,522; 5,232,925; 5,260,445; 5,814,647; 5,902,726;5,994,554; 6,294,580; 6,306,854; 6,498,174; 6,506,781; 6,541,492;6,552,055; 6,579,893; 6,586,455, 6,660,716, 6,673,823; 6,680,387;6,768,008; 6,787,551; 6,849,741; 6,878,749; 6,958,355; 6,960,604;7,022,722; and U.S. Applications 20030130306, 20030134885, 20030109579,20030109560, 20030088103, 20030087902, 20030096846, 20030092697,20030087935, 20030082631, 20030078288, 20030073862, 20030055265,20030045553, 1 20020169192, 20020165282, 20020160997, 20020128260,20020103188, 20020082292, 20030092736, 20030069275, 20020151569, and20030064935. The disclosures of these publications are incorporatedherein by reference in their entireties, especially with respect to thePPARγagonists disclosed therein, which may be employed in the methodsdescribed herein.

The inflammatory or damaging insult used to induce hyper iNOS expressionfrom the myeloid cells treated with or administered a PPARγ agonist andan IL-6/STAT3 signaling pathway antagonist can include any inflammatoryor damaging insult that elicits an inflammatory response from themyeloid cells. For example, the inflammatory or damaging insult caninclude or be administered via trauma, physical stress, contact with achemical, contact with biological agent, and/or exposure to radiation.Chemical insult in order to promote iNOS expression can include, forexample, local or systemic exposure or contact with thioglycolate(peritoneum), bleomycin (lung), and CCl₄ (liver). Biological insult caninclude local or systemic exposure to an endotoxin, such asLipopolysaccharides (LPS), a complement protein, ligands for acomplement receptor, or an anti-CD11b antibody. Radiation can includelocal or systemic exposure to therapy or interventional radiationtherapy or phototherapy, including but not limited to UV phototherapy,laser therapy, RF ablation therapy, external beam radiotherapy,brachytherapy, unsealed source radiotherapy, particle therapy, andradioisotope therapy. In certain aspects of the application, a physicalinsult to a subject's skin can include exposure to UV phototherapy orexternal beam radiotherapy.

It will be appreciated that the methods of the application are notlimited to above-identified PPARγ agonists, IL-6/STAT3 signaling pathwayantagonists, and inflammatory and damaging insults and that otheridentified PPARγ agonists, IL-6/STAT3 signaling pathway antagonists, andinflammatory and damaging insults can also be used.

It is contemplated that the accumulation of HiNOS cells in a region ofinterest and the hyper expression of iNOS in that region, results inlocal tissue destruction. Without being bound by theory, it is believedthat a product of iNOS (e.g., superoxide, a precursor to peroxynitrite)can mediate the destruction of pathogens, and unwanted cells and tissue.In an embodiment of the application, an inflammatory or damaging insultcan be administered directly to a site of infection or to a site ofunwanted or diseased tissue previous, during, and/or followingadministration of the PPARγ agonist and an IL-6/STAT3 signaling pathwayantagonist to myeloid cells, which are treated in vivo or administeredto the subject after in vitro administration of the PPARγ agonist and anIL-6/STAT3 signaling pathway antagonist.

Another embodiment of the application therefore relates to a method ofmediating local destruction of tissue in a subject. The method includesadministering an amount of a PPARγ agonist and IL-6/STAT3 signalingpathway antagonist to myeloid cells (e.g., macrophages) of the subjecteffective to substantially inhibit STAT3 activation in myeloid cells inthe subject. Prior to, during, and/or following administration of the aPPARγagonist and IL-6/STAT3 signaling pathway antagonist, the tissue canbe subjected to an inflammatory or damaging insult to induce hyper iNOSexpression in the myeloid cells that are in or about the periphery ofthe tissue and mediate local destruction of the tissue.

In one embodiment, the method can be used to mediate local destructionof neoplastic tissue. In the method, a therapeutically effective amountof a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonist can beadministered to myeloid cells of the subject, either systemically orlocally, e.g., directly to or about the periphery of a subject'sneoplastic tissue, to substantially inhibit STAT3 activation in themyeloid cells. An inflammatory or damaging insult can then beadministered to the neoplastic tissue to induce hyper iNOS expression ofthe myeloid cells that are in or about the periphery of the neoplastictissue and promote local destruction of the neoplastic tissue.

Neoplastic tissues that can be treated using the method of theapplication can include cancers or tumors including (but not limitedto): leukemias, such as but not limited to, acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemias, such as, myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemiasand myelodysplastic syndrome; chronic leukemias, such as but not limitedto, chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone and connectivetissue sarcomas such as but not limited to bone sarcoma, osteosarcoma,chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods of the application can be used in the treatmentof a variety of cancers or other abnormal proliferative diseases,including (but not limited to) the following: carcinoma, including thatof the bladder, breast, prostate, rectal, colon, kidney, liver, lung,ovary, pancreas, stomach, cervix, thyroid and skin; including squamouscell carcinoma; hematopoietic tumors of lymphoid lineage, includingleukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoictictumors of myeloid lineage, including acute and chronic myelogenousleukemias and promyclocytic leukemia; tumors of mesenchymal origin,including fibrosarcoma and rhabdomyoscarcoma; other tumors, includingmelanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumorsof the central and peripheral nervous system, including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer and teratocarcinoma. It is alsocontemplated that cancers caused by aberrations in apoptosis would alsobe treated by the methods and compositions of the invention. Suchcancers may include but not be limited to follicular lymphomas, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated in the skin, lung, colon, rectum, breast,prostate, bladder, kidney, pancreas, ovary, or uterus. In other specificembodiments, sarcoma, melanoma, small lung carcinoma, or leukemia istreated.

In some embodiments, the cancer is malignant. In other embodiments, thedisorder to be treated is a pre-cancerous condition. In specificembodiments, the pre-cancerous condition is high-grade prostaticintraepithelial neoplasia (PIN), fibroadenoma of the breast, orfibrocystic disease.

In certain embodiments, the inflammatory or damaging insult can bedelivered to cancer cells by site-specific means. Cell-type-specificdelivery can be provided by conjugating an inflammatory molecule to atargeting molecule, for example, one that selectively binds to theaffected cells. Methods for targeting include conjugates, such as thosedescribed in U.S. Pat. No. 5,391,723. Targeting vehicles, such asliposomes, can be used to deliver a compound, for example, byencapsulating the compound in a liposome containing a cell-specifictargeting molecule. Methods for targeted delivery of compounds toparticular cell types are well-known to those skilled in the art.

In certain embodiments, therapy by administration of one or more thePPARγ agonist and the IL-6/STAT3 signaling pathway antagonist can becombined with the administration of one or more therapies such as, butnot limited to, chemotherapies, radiation therapies, hormonal therapies,and/or biological therapies/immunotherapies. Prophylactic/therapeuticagents include, but are not limited to, proteinaceous molecules,including, but not limited to, peptides, polypeptides, proteins,including post-translationally modified proteins, antibodies etc.; orsmall molecules (less than 1000 daltons), inorganic or organiccompounds; or nucleic acid molecules including, but not limited to,double-stranded or single-stranded DNA, or double-stranded orsingle-stranded RNA, as well as triple helix nucleic acid molecules.Prophylactic/therapeutic agents can be derived from any known organism(including, but not limited to, animals, plants, bacteria, fungi, andprotista, or viruses) or from a library of synthetic molecules.

In certain embodiments, the neoplastic tissue can be cutaneous melanomaand non-melanoma skin cancer tissue. The method can include melanoma andnon-melanoma skin cancer ablation, wherein the subject is administered atherapeutically effective amount of a PPARγ agonist and an IL-6/STAT3signaling pathway antagonist before, during, or after surgical removalof a cancerous lesion in order to increase progression-free survivaltime.

In some aspects, the methods described herein are used in the treatmentof pathogenic infection. It is contemplated that increasing theexpression of iNOS by localized generation of HiNOS cells directly at orabout the periphery of a site of infection can inhibit a pathogenicinfection. For example, a therapeutically effective amount of a PPARγagonist and an IL-6/STAT3 signaling pathway antagonist can beadministered to the subject systemically or locally, e.g., directly toor about the periphery of pathogenic infection, in order to increaseiNOS expressing cells at the site to mediate the destruction of apathogenic organism.

It will be appreciated that the methods of application need not belimited to the treatment of pathological conditions or uses and can beused for cosmetic applications. For example, the methods describedherein can be used for removal of scar tissue in a subject, wherein atherapeutically effective amount of PPARγ agonist and IL-6/STAT3signaling pathway antagonist is administered to myeloid cells of thesubject in combination with an inflammatory or damaging insult (e.g., UVradiation) directly to or about the periphery of the subject's scartissue. In other examples, the methods described herein can be used forremoval of a tattoo of a subject, unwanted hair, and/or an actinickeratosis lesion wherein a therapeutically effective amount of a PPARγagonist and an IL-6/STAT3 signaling pathway antagonist is administeredto myeloid cells of the subject in combination with a inflammatory ordamaging insult (e.g., laser therapy or photorejuvenation) directly toor about the periphery of the tissue being treated (e.g., tattoo, hairfollicle, skin).

The methods contemplated herein can also be used for inhibiting normalwound repair in a subject. The method includes administering atherapeutically effective amount of a PPARγ agonist and an IL-6inhibitor to the subject. The therapeutically effective amount is theamount of PPARγ agonist and IL-6 inhibitor can substantially increaseHiNOS expressing cells at or about the periphery of the wound of thesubject. For example, the method may be used to inhibit adhesions in asubject (e.g., peritoneal adhesions) and scar formation (e.g., keloidformation, hypertrophic scarring, and striae).

Other embodiments of the application relate to pharmaceuticalcompositions including at least one PPARγ agonist and at least oneIL-6/STATS signaling pathway antagonist described above. Pharmaceuticalcompositions described herein will generally include an amount of PPARγagonists and IL-6/STAT3 signaling pathway antagonist admixed with anacceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980, incorporated herein by reference.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards. Upon formulation, the polypeptide orconjugate solutions will be administered in a manner compatible with thedosage formulation and in such amount as is therapeutically effective.

The dose, amount, and/or quantity of the pharmaceutical compositionsdescribed above which are administered to the subject can depend on thespecific IL-6/STAT3 signaling pathway antagonist(s) and PPARγ agonist(s)selected. It will be appreciated that the dosage amounts used willdepend on the potency of the specific PPARγ agonist and the therapeuticregimen employed.

In another aspect, the IL-6/STAT3 signaling pathway antagonist and PPARγagonist when administered in combination to subject can be administeredat an amount or dosage to achieve a therapeutic effect that issubstantially less (i.e., subtherapeutic dose or amount) than the amountor dose that would be required to achieve a therapeutic effect if eachcompound was administered alone. Co-administration of the IL-6/STAT3signaling pathway antagonist and PPARγ agonist to the subject can alsomitigate resistance to one single agent. Such resistance results eitherin the requirement for higher dosages of the drug and/or the renewedsymptoms.

Moreover, co-administration of IL-6/STAT3 signaling pathway antagonistand PPARγ agonist to the subject can mitigate toxicity and side effectsassociated with potentially administering a single agent at an amounteffective to achieve a therapeutic effect. For example, according to anFDA alert issued on May 21, 2007, therapeutic doses of the PPARγ agonistrosiglitazone, are associated with a substantially increased risk ofheart attack, and even higher risk of death from all cardiovasculardiseases. In addition, both rosiglitazone and pioglitazone have beensuspected of causing macular edema. Therefore, there is a practicalupper limit to the amount that a subject can receive. However, if two ormore agents are used in concert, the dosage of any single drug can belowered. This is beneficial to the patient since using lower levels oftherapeutic agents is generally safer for the patient. Additionally,cells are less likely to generate resistance to the combination of drugsas they are to a single drug. Thus, in some aspects of the presentinvention, the compositions described herein can be administered to asubject at a subtherapeutic level.

The method of the application is not limited by the order in which theagents are administered. In one embodiment, the agents are administeredsequentially. In another embodiment, the agents are administered as acombined formulation (e.g., a formulation comprising an IL-6/STAT3signaling pathway antagonist and a PPARγ agonist).

The agents described herein are not limited by the route ofadministration. For example, the pharmaceutical compositions may beadministered orally, intravenously, intraperitoneally, or by directintralesional injection. In some aspects of the present invention,pharmaceutical compositions may be administered directly to or about theperiphery of a lesion or wound by injection, for example, underbronchoscopy, endoscopy, intra-operativley (e.g., during surgery) or asan adjuvant therapy, or in the case of dermatological disorders (e.g.,skin cancer), for example, by direct application of creams or ointments.In certain aspects, one agent is administered by one route, while thesecond agent is administered by a second route.

The IL-6/STAT3 signaling pathway antagonist and PPARγ agonist can beformulated for systemic administration and/or topical administration.Advantageously, the IL-6/STAT3 signaling pathway antagonist and PPARγagonist can be administered by local topical administration to a regionof interest, for example, the site of an unwanted tissue. Topicaladministration is desirable because a lower dosage can be administeredto the subject being treated to provide a therapeutically effectivebenefit. Additionally, administration of a lower topical dosage canmitigate adverse side-effects that may be associated with systemicadministration.

Topical formulations include those for delivery via the mouth (buccal)and through the skin such that at least one layer of skin (i.e., theepidermis, dermis, and/or subcutaneous layer) is contacted with anIL-6/STAT3 signaling pathway antagonist and PPARγ agonist. Topicaldelivery systems may be used to administer topical formulations of thepresent invention. Topical delivery systems can include, for example,transdermal patches containing an IL-6/STAT3 signaling pathwayantagonist and PPARγ agonist or derivative thereof to be administered.Delivery through the skin can further be achieved by iontophoresis orelectrotransport, if desired.

Formulations for topical administration in the mouth can include any oneor combination of: lozenges comprising an IL-6/STAT3 signaling pathwayantagonist and PPARγ agonist in a flavored basis, usually sucrose andacacia or tragacanth; pastilles comprising an IL-6/STAT3 signalingpathway antagonist and PPARγ agonist in an inert basis such as gelatinand glycerin or sucrose and acacia; and mouthwashes comprising anIL-6/STAT3 signaling pathway antagonist and PPARγ agonist to beadministered in a suitable liquid carrier.

Formulations for topical administration to the skin can includeointments, creams, gels, and pastes comprising IL-6/STAT3 signalingpathway antagonist and PPARγ agonist to be administered in apharmaceutically acceptable carrier. Topical formulations foradministration to the skin can include creams, ointments, and gels, forexample, and can be prepared using oleaginous or water-soluble ointmentbases, as is well known to those in the art. For example, theseformulations may include vegetable oils, animal fats, and morepreferably, semisolid hydrocarbons obtained from petroleum. Particularcomponents used may include white ointment, yellow ointment, cetylesters wax, oleic acid, olive oil, paraffin, petrolatum, whitepetrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin, and glyceryl monostearate. Variouswater-soluble ointment bases may also be used including, for example,glycol ethers and derivatives, polyethylene glycols, polyoxyl 40stearate, and polysorbates. Other examples of topical deliverycompositions that can be used for delivering the IL-6/STAT3 signalingpathway antagonist and/or PPARγ agonist include polycosanol combinationsand nanoparticulate aersol formulations described, for example, in U.S.Pat. Nos. 7,521,068 and 7,763,278.

Another aspect of the application relates to a method of generating ananimal model of chronic delayed wound healing. The method includes thein vivo generation of HiNOS cells in an animal and the administration ofa non-lethal wound. In some aspects, the animal is an IL-6^(−/−) animalthat has been wounded and administered a PPARγ agonist. However, it iscontemplated that an IL-6/STAT3 signaling pathway antagonist describedherein can be administered to an IL-6^(+/+) animal.

In one exemplary embodiment, a delayed wound healing animal model isgenerated by shaving and depilating 6-8 week old female IL-6^(−/−) micea minimum of 3 days prior to experimentation. On day 0, the mice receive3×6 mm punch biopsies on their dorsal side. Mice are then subjected to alow dose of ultraviolet radiation (e.g., 72 mJ/cm² UVB), and a topicalPPARγ agonist is applied in a petrolatum. From there, topical PPARγagonist is applied daily after wound size measurements are obtained.

In some aspects of the invention, an animal model is characterized by anincrease in time for wound healing of at least about 50%, at least about100%, at least about 150%, or at least about 200% compared to a control.An animal model can also be characterized by enlargement of a wound,failure to initiate wound healing with sustained enlarged wounds, anddelayed complete re-epithieliazation compared to a control.

A model can be used to determine interventions and therapies to treatboth acute and chronic delayed wounds. Therefore, the generation ofmodel of delayed wound healing provides the means to study 1) mechanismsthat delay wound repair and 2) clinically applicable therapies toincrease the rate of healing.

The animal model can be employed in testing the efficacy of drugcompounds aimed at treating delayed wound healing. For example, theanimal model of the can be used in a method of identifying agents thatstimulate wound repair and the healing of damaged tissue. The methodincludes administering a test compound to a mammal either before orafter wound or damage is administered. A test compound can then beadministered (e.g., via topical administration or injection). In oneaspect, a test compound that causes a significant initiation of woundhealing, decrease in the time of wound repair, and/orre-epithieliazation compared to a control is indicative of an effectiveagent.

In another example, the animal model can be used in a method ofidentifying an agent that inhibits unwanted wound healing. The methodincludes administering to a mammal a test compound either before orafter wound or damage is administered. A test compound that causessignificant delay or increase in the time of wound repair is indicativeof an effective agent.

While hyper iNOS expression from HiNOS cells can be advantageous in sometherapeutic applications, excessive nitric oxide (NO) production viainduction of iNOS can lead to unwanted local tissue damage and has beenimplicated in numerous destructive inflammatory diseases, a delay inwound healing, and local destruction of tissue (destruction of hairfollicles). Delayed wound healing can result from a sustainedinflammatory state at the wound site. Thus, it is further contemplatedby the application that agents that inhibit iNOS expression in a subjectcan be used to treat inflammatory disease and/or promote wound healingin a subject. Of particular relevance are subjects that have reducedIL-6 expression, hereditarily or as the result of therapies that causeimpaired IL-6 expression (e.g., statin therapy) and/or subjects thatalso are being treated with PPARγ agonists (e.g., diabetics). Suchsubjects can be prone to delayed wound healing. Such delayed woundhealing is commonly observed in diabetic ulcers.

Therefore, in another aspect, a method of promoting wound healing in asubject that has impaired IL-6 expression and/or a reduced STATSactivation in myeloid cells (e.g., a subject undergoing statin and PPARγagonist therapy). The method includes administering to the wound orabout the periphery of the wound a therapeutically effective amount ofIL-6 wherein a therapeutically effective amount is the amount of IL-6 tosubstantially decrease iNOS expression myeloid cells in a subject. Insome aspects, the IL-6 can include recombinant IL-6 (rIL-6).

The wound treated by the method and/or compositions described herein caninclude any injury to any portion of the body of a subject (e.g.,internal wound or external wound) including: acute conditions or wounds,such as thermal burns, chemical burns, radiation burns, burns caused byexcess exposure to ultraviolet radiation (e.g., sunburn); damage tobodily tissues, such as the perineum as a result of labor andchildbirth; injuries sustained during medical procedures, such asepisiotomies; trauma-induced injuries, such as cuts, incisions,excoriations, injuries sustained as result of accidents, ulcers, such aspressure ulcers, diabetic ulcers, plaster ulcers, and decubitus ulcer,post-surgical injuries. The wound can also include chronic conditions orwounds, such as pressure sores, bedsores, conditions related to diabetesand poor circulation, and all types of acne. In addition, the wound caninclude dermatitis, such as impetigo, intertrigo, folliculitis andeczema, wounds following dental surgery; periodontal disease; tumorassociated wounds. In one aspect of the invention, the wound can berelated to sarcoidosis or cirrhosis in a subject.

A method of promoting wound healing in accordance with some aspects ofthe application can include restoring wound healing in a subject thathas impaired IL-6 expression and/or a reduced STAT3 activation inmyeloid cells where there has been a significant delay in wound healing.For example, it is often desirable to promote or increase the rate ofhealing in the case of both chronic wounds (such as diabetic, venous),acute (such as burns, penetrative injuries, or even wounds resultingfrom elective surgery), and for healing compromised individuals (such asimmunodeficencies and the elderly). In all examples, the wounds, and adelay in healing of the wounds, can in the worst-case lead to death, butin general severely decrease the quality of life. It will be appreciatedthat the present application is not limited to the preceding wounds orinjuries and that other wounds or tissue injuries whether acute and/orchronic can be treated by the compositions and methods of the presentinvention.

In addition, an iNOS inhibitor can be administered alone or incombination with IL-6 for the promotion of wound healing in a subject.An iNOS inhibitor for use in the present invention can include thehighly selective iNOS inhibitor, 1400 W (see Garvey et al. (1997) 1400 Wis a Slow, Tight Binding, and Highly Selective Inhibitor of InducibleNitric Oxide Synthase In vivo and In vitro., J Biol. Chem. February 21;272(8):4959-63).

In other aspects, a TNF-α inhibitor can be administered to a subjectalone or in combination with IL-6 in order to promote wound healing in asubject. A TNF-α inhibitor for use in the present invention can includea monoclonal antibody such as infliximab, adalimumab, certolizumabpegol, and golimumab, a circulating receptor fusion protein such asetanercept, or simple molecules such as xanthine derivatives (e.g.,pentoxifylline) and bupropion.

Therefore, in another aspect a method of promoting wound healing in asubject that has impaired IL-6 expression and/or a reduced STAT3activation in myeloid cells is provided. The method includesadministering to the subject a therapeutically effective amount of IL-6,TNF-α inhibitor, and/or iNOS inhibitor to the subject wherein atherapeutically effective amount is the amount of IL-6, TNF-α inhibitor,and/or an iNOS inhibitor to substantially decrease iNOS expression in asubject.

In some aspects, the IL-6, TNF-α inhibitor, and/or iNOS inhibitor can beadministered to a subject systemically. In some aspects, the anti IL-6,TNF-α inhibitor, and/or iNOS inhibitor can be administered directly toor about the periphery of a wound. In one example, the period of timethat the IL-6, TNF-α inhibitor, and/or iNOS inhibitor is administered tothe wound and/or proximate the wound can comprise from about onset ofthe wound and/or tissue injury to about days, weeks, or months aftertissue injury.

The present application further relates to pharmaceutical compositionsincluding IL-6, TNF-α inhibitor, and/or iNOS inhibitor described above.Examples of pharmaceutical compositions in accordance with the inventionwill generally include an amount of an IL-6, TNF-α inhibitor, and/oriNOS inhibitor admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a range of finalconcentrations, depending on the intended use.

In another aspect of the invention, IL-6, TNF-α inhibitor, and/or iNOSinhibitor and pharmaceutical compositions thereof can be provided inand/or on a substrate, solid support, and/or wound dressing for deliveryof the IL-6, TNF-α inhibitor, and/or iNOS inhibitor to the wound. Asused herein, the term “substrate,” or “solid support” and “wounddressing” refer broadly to any substrate when prepared for, and appliedto, a wound for protection, absorbance, drainage, etc. The presentinvention may include any one of the numerous types of substrates and/orbackings that are commercially available, including films (e.g.,polyurethane films), hydrocolloids (hydrophilic colloidal particlesbound to polyurethane foam), hydrogels (cross-linked polymers containingabout at least 60% water), foams (hydrophilic or hydrophobic), calciumalginates (non-woven composites of fibers from calcium alginate), andcellophane (cellulose with a plasticizer). The shape and size of a woundmay be determined and the wound dressing customized for the exact sitebased on the measurements provided for the wound. As wound sites canvary in terms of mechanical strength, thickness, sensitivity, etc., thesubstrate can be molded to specifically address the mechanical and/orother needs of the site. For example, the thickness of the substrate maybe minimized for locations that are highly innervated, e.g., thefingertips. Other wound sites, e.g., fingers, ankles, knees, elbows andthe like, may be exposed to higher mechanical stress and requiremultiple layers of the substrate.

Pharmaceutical compositions described herein can also be provided in oron a surface of a medical device used to treat an internal and/orexternal wound. The medical device can comprise any instrument,implement, machine, contrivance, implant, or other similar or relatedarticle, including a component or part, or accessory, which is, forexample, recognized in the official U.S. National Formulary, the U.S.Pharmacopoeia, or any supplement thereof; is intended for use in thediagnosis of disease or other conditions, or in the cure, mitigation,treatment, or prevention of disease, in humans or in other animals; or,is intended to affect the structure or any function of the body ofhumans or other animals, and which does not achieve any of its primaryintended purposes through chemical action within or on the body of manor other animals, and which is not dependent upon being metabolized forthe achievement of any of its primary intended purposes.

The following examples are included to demonstrate an embodiment of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples, which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 IL-6 and PPARγ Agonists Generate Hyper-InflammatoryMacrophages Leading to Tissue Destruction

We found that treatment of myeloid cells (e.g., macrophages) withrosiglitazone in the absence of IL-6 in vitro and in vivo results in 1)synergistic hyper-inflammatory responses, 2) generation ofpro-inflammatory, hyper-iNOS and TNF-γ expressing macrophages, and 3)drastic macrophage-mediated de novo tissue destruction. Our datademonstrate that hyperinflammatory macrophages are induced in severalmodel systems suggesting that these findings may be clinically relevantto a large number of patients, given the common concurrence of relativestates of IL-6 inhibition with PPARγ activation induced either bypharmacologic agonists or by endogenous ligands generated duringinflammation and oxidant stress.

Methods Mice

Pathogen-free, 6-8 week old female C57BL/6J wildtype mice were obtainedfrom Jackson Laboratories (Bar Harbor, Me.). IL6^(−/−) mice wereoriginally obtained from Jackson Laboratories (Bar Harbor, Me.) andmaintained for subsequent generations at the Animal Resource Center atCWRU (Cleveland, Ohio).

Immunofluorescence

Macrophages were defined as cells that stained positive for F4/80 andCD11b. For immunofluorescence staining of skin, 8 μm sections of frozentissue was sectioned and fixed in 4° C. 100% acetone for 10 minutes. Fori.p. and bone marrow derived macrophages, cells were washed with PBS andthen fixed with 4% paraformaldehyde for 10 min at room temperature.After washing twice with PBS, cells were blocked in a solution of PBScontaining 3% BSA, for 30 min at room temperature. Forimmunofluorescence of lung tissue, 5 μm sections were fixed in formalin,embedded in paraffin, and deparaffinized in xylene and ethanol. Primaryand secondary antibodies were diluted as inidcated in 1×PBS. Primary andsecondary antibodies were typically applied 90 minutes. Cells werewashed three times with PBS between primary and secondary staining. Thefollowing antibodies were used for our analysis: anti-mouse F4/80Antigen Alexa-488 BM8 (eBioscience, 1:100); Rat monoclonal [M1/70] toCD11b APC/Cy7 (abcam, 1:20); rabbit anti-iNOS/iNOS II, NT (Millipore,1:50). A secondary antibody conjugated to Alexa-405 was obtained fromInvitrogen.

RT-PCR

For RT-PCR analysis, RNA was isolated using Trizol (Invitrogen)following the manufacturer's instructions, and was reverse-transcribedand quantified using Applied Biosystems TaqMan® RNA-to-CT™ 1-Step Kit,TaqMan® Gene Expression Assays for 18 s RNA, iNOS II, TNF-γ, and IL-1β,and Step-One™ System. Cycle time, temperature, and number were based onApplied Biosystems recommendations.

Wound Preparation, Treatment and Measurement

Skin was wounded with some modifications. Mice were treated with 3-6 mmexcisional wounds, one midline and two on each side of the midline. AtDay 0, wounds were measured lengthwise and widthwise, treated with 1 MEDUVB, and treated topically with a 1% solution of rosiglitazone in 10 gAquaphor. Wounds were measured and treated with topical rosiglitazonesolution daily until healed (loss of serum crust andreepithelialization).

Lung Treatment and Preparation

Animals were anesthetized with avertin (250 mg/Kg, i.p., and taped to asurgical board). The neck area was thoroughly cleaned with betadinescrub and alcohol. A small midline skin incision (approx. 0.8 cm) wasmade in the neck and the trachea was exposed. Using a 27 gauge needleinserted between cartilaginous rings, 30 μl of phosphate-buffered salinecontaining 0.075 units of bleomycin was injected. The skin was closedwith surgical glue (nexaband).

Alveolar Macrophages

Mice were injected intraperitoneally (i.p.) with a lethal dose ofavertin (1 ml of 20 g/ml solution). Bronchoalveolar lavage (BAL) wasperformed by inserting a cannula through a cut in the trachea into thebronchi and infusing 3×0.5 ml aliquots of PBS. The BAL fluid sample wasrecovered by aspirating the liquid with a syringe; we recovered 80%±15%of instilled fluid volume. Macrophages were harvested following a spindown of the BAL fluid 25.

Peritoneal Macrophages

Mice were injected i.p. with 2 ml of thioglycollate and sacrificed 24hours later mice by cervical dislocation. Lavage was performed byinfusing 5 ml of 40 C DPBS into the peritoneal cavity. The fluid sampleis recovered by aspirating the liquid with a syringe. 80%±21% ofinstilled fluid volume is recovered. Macrophages were harvestedfollowing centrifugation of the BAL fluid.

Bone Marrow Derived Macrophages

Briefly, bone marrow cells were flushed aseptically from the dissectedfemurs of mice with PBS. The cells were cultured on 25 cm plates in DMEMcontaining 15% Fetal Calf Serum, 1% pen strep, 1% L-glutamine, 4.5 g/Lglucose, and 20 μg/L MCSF. After 6 days, the media was changed to removeany cellular debris and non-adherent cells. The following day (day 7),the cells were carefully removed using lidocaine (4 mg/mL) and 10 mMEDTA in PBS at pH 7.5, and dispensed into 6 well plates at aconcentration of 2×10⁶ cells/well. Prior to experimentation, media wasreplaced with MCSF-free media containing 5 mM L-arginine.

Western Blot

NE-PER kit (Thermo Scientific) was used to extract nuclear andcytoplasmic protein from peritoneal macrophages. Protein samples wereprepared under reduced conditions and western blot analysis wasperformed using a standard protocol (Invitrogen). Stat3 (79D7) RabbitmAb, NF— xB p65, β-actin (13E5) Rabbit mAb (Cell Signaling Technology,Danvers, MA) were used for western blot evaluation. Densiometry analyzedby Biorad VersaDoc imaging system.

Antibodies and Reagents

Anti-TNF-α (abcam), rIL-6 (R&D Systems), anti-CD11b (AbD serotec) wereall given i.p at a 1 μg/1 g body weight. 1400 W (Sigma) was given at a10 μg/g dose. All other reagents were purchased from sigma.

Statistical Analysis

Data presented as means±s.e.m. P values were calculated using atwo-tailed Student's t test for two samples of unequal variance.Statistical significance is indicated by an asterisk, and P valuesreported in figure legends.

Results

PPARγ Ligands Drive Hyper-Expression of iNOS and TNF-α in the Absence ofIL-6

To investigate the effects of PPARγ activation in macrophages, weisolated primary bone marrow derived macrophages (BMDMs) from IL6^(−/−)or matched wildtype C57BL/6 mice. As expected, in vitro stimulation ofwildtype and IL6^(−/−) BMDMs with LPS resulted in a modest upregulationof iNOS and TNF-α mRNA. However, pre-treatment of IL6^(−/−) BMDMs withrosiglitazone followed by LPS stimulation produced striking increases inboth iNOS and TNF-α expression (FIG. 2A, B). To determine whether the invitro response has in vivo significance, we utilized the model ofthioglycollate-induced peritonitis to elicit a robust macrophageresponse in the peritoneum. Macrophages isolated from wildtype andIL6^(−/−) mice subjected to peritoneal injury with or without in vivorosiglitazone treatment were harvested and analyzed ex vivo. Similar tothe in vitro results, rosiglitazone treated IL6^(−/−) peritonealmacrophages expressed substantially higher levels of iNOS and TNF-α(FIG. 3A, B). Confocal immunofluorescence microscopy using anti-CD11band anti-F4/80 confined that the peritoneal cells were monocytes andmacrophages (data not shown). To determine if generation of thishyper-inflammatory macrophage phenotype is driven by PPARγ and not anoff-target effect of rosiglitazone, we utilized pioglitazone, anotherPPARγ agonist, and observed no significant difference between the twoPPARγ agonists on iNOS or TNF-α expression (FIG. 4A, B). To test if thepro-inflammatory effects were dependent on PPARγ signaling, IL6^(−/−)mice were treated with either rosiglitazone or GW9662, a compound thatbinds the same receptor but does not signal through PPARγ. Nosignificant increase in iNOS was observed in GW9662 treated micedemonstrating the importance of intact PPARγ signaling (FIG. 5). Todetermine if these in vivo findings were applicable in other organs, weutilized a lung injury model induced by bleomycin, a chemotherapeuticknown to induce fibrosis and inflammation. Bronchoalveolar lavage (BAL)fluid was collected from mice 24 hours following treatment with eitherintratracheal bleomycin or sham intratracheal procedure with or withouti.p. rosiglitazone treatment. As has been previously reported,macrophages are the predominant cell in BAL fluid (data not shown).Similar to the in vitro data and in vivo peritonitis data, bleomycininjury combined with IL-6 deficiency and systemic PPARγ agonisttreatment induced significant expression of iNOS and TNF-γ expression inalveolar monocytes/macrophages (FIG. 6A, B). These data demonstrate thatorgan injury combined with PPARγ activation and IL-6 deficiency leads tohyperinduction of iNOS and TNF-γ mRNA expression in infiltratingmacrophages. Modest increases in iNOS and TNF-γ expression from micetreated with a sham procedure or bleomycin alone reflects inflammationdue to tracheotomy-associated injury. To determine a potential mechanismfor hyper-induction of iNOS under our treatment conditions, we examinedtranscription factors involved in the cooperative regulation of iNOS,specifically nuclear factor κB (NF-κB), and signal transducer andactivator of transcription 3 (STAT3). NF-κB is a potent activator ofiNOS expression upregulated under inflammatory conditions. STAT3 hasbeen previously demonstrated to inhibit NF-kB-mediated transactivationof iNOS, resulting in decreased iNOS expression. Additionally, PPARγligands have been shown to decrease STAT3 phosphorylation, a criticalstep for nuclear translocation of the transcription factor. Given this,we hypothesized that in our experimental condition, STAT3-mediatedsuppression of NF-κB transactivation of iNOS is diminished, resulting inhyper-induction of iNOS. Thioglycollate-elicited macrophages harvestedfrom both IL6^(−/−) and WT mice were treated with rosiglitazone ex vivofollowed by LPS stimulation. Timepoint analysis of nuclear NF-κB p65 andSTAT3 fold change from 30 to 60 minutes show a striking increase inNF-kB p65 with concomitant decrease of STAT3 in IL6−/− macrophagestreated with rosiglitazone and LPS (FIG. 7A, B). Interestingly, thecombination of rosiglitazone and a stimulatory agent, in the absence ofIL-6, is the treatment condition in which we observe hyper-induction ofiNOS (FIGS. 2-7). Reconstitution of IL6−/− macrophages with recombinantIL-6 (rIL6) restores STAT3 and NF-kB levels similar to wildtypeconditions (FIG. 7B).

Hyper Inflammatory Macrophage Generation In Situ

Next, we investigated the effects of PPARγ activity on macrophages insitu using models of pneumonitis and dermatitis. As was observed in BALmacrophages (FIG. 8A,B), RTPCR of RNA from whole lungs revealedincreased iNOS and TNF-α expression when treated with a PPARγ agonist inIL6^(−/−) mice, starting at 6 hours (data not shown) and peaking at 24hours (FIG. 8A,B). No increases in iNOS or TNF-γ were observed inwildtype counterparts. To determine the cellular source of whole lungiNOS expression, lung sections were stained with antibodies againstmonocytes/macrophage markers F4/80 and CD11b, and iNOS. Althoughconfocal microscopy demonstrated colocalization of CD11b and F4/80,little iNOS staining was observed in wildtype animals treated withbleomycin and rosiglitazone (FIG. 9A). IL6^(−/−) mice treated in anidentical manner displayed intense staining of iNOS which colocalizedwith CD11b⁺F4/80⁺ macrophages (FIG. 9B). Little iNOS staining wasobserved in either wildtype or IL6^(−/−) mice treated with bleomycin orrosiglitazone alone (FIG. 18A-F). In the skin, IL6^(−/−) and wildtypemice were subjected to excisional biopsies on dorsal skin followed by asingle dose of 72 mJ/cm² UVB and topical application of rosiglitazone.This dose of UVB has been previously shown to recruit monocytes andmacrophages into the skin. In wildtype mice, rosiglitazone or UVB alone,or in combination, did not result in a significant increase of iNOS andTNF-α expression. A similar modest induction of iNOS and TNF-α mRNA wasobserved in IL6^(−/−) mice, except in the combined wounding, UVB, androsiglitazone treatment condition. This combination resulted in a20-fold and 13-fold increase in iNOS and TNF-α mRNA expression at 24hours, respectively (FIG. 10 A, B). To determine the cellular source ofiNOS, skin sections were stained for F4/80, CD11b, and iNOS. Confocalmicroscopy demonstrated low iNOS levels in wildtype animals (FIG. 11A),yet robust iNOS staining colocalized with CD11b+F4/80− monocytes andCD11b⁺F4/80⁺ macrophages in IL6^(−/−) mice that were wounded prior totreatment with UVB and rosiglitazone (FIG. 11B). Taken together, the invitro and in vivo results demonstrate that the presence of a PPARγagonist in the absence of IL-6 generates hyper-inflammatory macrophagesin multiple systems.

Hyper Inflammatory Macrophage Generation Leads to Destruction of Tissue

After observing significant increases in macrophage-derived inflammatorymediators and cytokines in our wound model, we hypothesized thatincreased iNOS and TNF-α expression would result in tissue destruction.Skin isolated from IL6^(−/−) mice following the combination ofUVB/rosiglitazone/wound treatment (“the inflammatory wound model”)displayed a dense infiltrate of mononuclear cells by 9 hours postwounding compared to controls as illustrated by H&E staining (FIG.12A,B). Confocal microscopy demonstrated the presence of CD11b⁺andF4/80⁺ cells in both IL6^(−/−) and wildtype mice (FIG. 12C, D).Surrounding wound skin was harvested at the indicated time points andanalyzed for iNOS and TNF-α expression. Wildtype mice displayed minimalincreases in iNOS and TNF-α. In contrast, iNOS and TNF-α weresubstantially upregulated in IL6^(−/−) mice as early as 9 hours whichcoincides with the entry of monocytes and macrophages into the skin(FIG. 13A, B). To determine the cellular specificity of iNOS, otherleukocytes such as neutrophils were also examined. Corresponding tissuesections were stained for the neutrophil marker GR1 (Ly6g) and iNOS. Nocolocalization of iNOS with neutrophils was observed (FIG. 14A).

To verify that monocytes/macrophages were responsible for the excessinflammation and tissue destruction, we used clodronate liposomes whichare selectively toxic to phagocytic cells to depletemonocytes/macrophages in the surrounding wound tissue. IL6^(−/−) micewere subjected to the inflammatory wound model followed by intradermalinjections of either clodronate or PBS control liposomes in thesurrounding wound areas. After 24 hours wounds from IL6^(−/−) micetreated with PBS control liposomes exhibited dermal edema composed of adense infiltrate of inflammatory cells in the dermis and subcutis. At 48hours the skin exhibited epidermal necrosis and superficial dermalnecrosis (FIG. 14B). In contrast, wounds from IL6^(−/−) mice treatedwith clodronate liposomes resulted in normal appearing tissue withpreservation of the epidermis and dermis, and a sparse mononuclear cellinfiltrate in the dermis and subcutis (FIG. 14C). We thus conclude thatin the absence of IL-6, PPARγ ligands drive macrophages towards a highiNOS and TNF-α hyper inflammatory state propagating inflammation andtissue destruction.

Hyper-Inflammatory Macrophage Generation Leads to a Delay in WoundHealing

Next we asked whether the increased macrophage-derived inflammatoryfactors would affect the duration of wound healing. IL6^(−/−) andwildtype mice were subjected to the previously described inflammatorywound protocol. Wounds from each mouse were measured daily and woundhealing curves were generated based on the percentage of the initialtotal wound area. Wounds in IL6^(−/−) mice topically treated withrosiglitazone drastically expanded compared to wildtype animals withcomparable initial wound size at day 0 (FIG. 15A). By day 3, wounds inIL6^(−/−) mice show visible inflammation in the intact skin between thewound sites and little contraction compared to wildtype. By day 9, thewounds of wildtype mice had progressed towards resolution, while thewounds of IL6^(−/−) mice remained enlarged above baseline (FIG. 15B).Strikingly, wound closure was delayed by 8 days in IL6^(−/−) micecompared to wildtype. Mice given daily i.p injections of rosiglitazonealso demonstrated wound expansion (data not shown), indicating that theroute of drug administration was not critical for subsequentmacrophage-mediated destruction. Wound closure was unaffected bytreatment with rosiglitazone alone, UVB alone, or vehicle alone (FIG.19A-D). To confirm the criticality of IL-6 in this model, we testedwhether reconstitution with rIL-6 in IL6^(−/−) animals would restorenormal wound healing. Wounds of rIL-6 reconstituted mice demonstratenormal wound healing with resolution by day 11, similar to wildtype(FIG. 15 b).

To further confirm the role of macrophages in mediating tissuedestruction, we tested whether blocking macrophage entry into the skinusing an antibody against CD11b would 10 restore tissue morphology.Anti-CD11b treatment has been previously shown to prevent infiltrationof monocytic cells elicited by UVB into the skin35. IL6^(−/−) mice weresubjected to the wound treatment along with either i.p. anti-CD11b. Skinwounds harvested at 24 hours in IL6^(−/−) mice treated with isotypecontrol exhibit epidermal necrosis, superficial dermal necrosis, dermaledema, and a dense infiltrate of inflammatory cells in the subcutis. Incontrast, wounds of IL6^(−/−) mice treated with i.p. injections ofanti-CD11b had intact epidermis and dermis with a sparse mononuclearinfiltrate in the subcutis (FIG. 15C). Confocal microscopy confirmednumerous CD11b⁺and F4/80⁺ cells in skin harvested 24 hours afterwounding from IL6^(−/−) mice injected with isotype control in contrastto sparse staining in skin of mice treated with anti-CD11b (FIG. 15D).Expression levels of iNOS and TNF-α were substantially lower in theIL6^(−/−) mice injected with anti-CD11b versus isotype controldemonstrating the role of macrophages in expression of theseinflammatory mediators (FIG. 16 A, B). To further test the roles ofmacrophages in delayed wound healing, we generated a wound healing curvefor IL6^(−/−) mice subjected to inflammatory wound treatment andinjected i.p. with anti-CD11b. Wounds from mice injected with anti-CD11bremained above baseline until day 5 and were fully healed by day 13. Incontrast, wounds of mice injected with CD11b isotype control remainedabove baseline until day 7, and did not heal until day 21 (FIG. 17A).Lastly, to demonstrate the roles of iNOS and TNF-α in our inflammatorywound model, we generated wound healing curves for IL6^(−/−) micesubjected to inflammatory wound treatment and a single i.p. injectionwith either 1400 W (a specific iNOS inhibitor) or a single injection ofanti-TNF-α 1 hour prior to wounding. The wounds from animals treatedwith 1400 W display a normal healing pattern, healing by day 14,compared to day 23 for PBS-injected mice (FIG. 17B). Lastly, wounds fromIL6^(−/−) mice treated with anti-TNF-α antibody regain a normal healingpattern and heal by day 12, compared to day 23 for isotype controlinjected mice (FIG. 17C).

Example 2 Combination of Statin and PPAR-γ Agonist Generates ParadoxicalHyper-Inflammation and Tissue Destruction

We found that PPAR-γ agonist rosiglitazone, its presence in the absenceof IL-6 paradoxically drives macrophages to express super-elevatedlevels of iNOS resulting in tissue destruction and delayed woundhealing. We were interested in investigating the interaction of statinswith rosiglitazone, because statins have been shown to reduce IL-6 andclinically, these medications are often co-prescribed in type IIdiabetics for cardiovascular disease prevention. We found that that LPSstimulation of macrophages pre-treated with mevastatin and rosiglitazoneresults in an 18.3-fold increase in iNOS expression compared to LPSalone (n=3, p=0.002) (FIG. 20). In vivo studies demonstrate that i.p.injection of simvastatin to mice prior to wounding, followed by dailytopical rosiglitazone administration, results in tissue destruction anda nine day delay in wound healing (n=3) (FIG. 21). We found that themechanism of increased iNOS following treatment is due to a decreasedability of STAT3 to transrepress NF-κB activity in the nucleus,resulting in unregulated iNOS expression. LPS stimulation of statin androsiglitazone pretreated macrophages results in upregulation of SOCS3 inthe cytoplasm followed by a decrease in STAT3 with a concomitantincrease in NF-κB accumulation in the nucleus (n=2). In summary, wedemonstrate that the presence of two medications with knownanti-inflammatory properties, rosiglitazone and mevastatin, results in aparadoxical increase in inflammation and a tissue destructive phenotype.

Example 3 Human HiNOS Generation with Combination of Statin and PPAR-γAgonist

FIG. 22 illustrates a graph showing iNOS expression in human macrophagestreated with statin, anti-CD11b, and PPAR-γ agonist. We unexpectedlyfound that human macrophages treated with a statin and PPAR-γ agonistexhibit over express iNOS compared to controls and human macrophagestreated with statin alone and PPAR-γ alone.

Monocyte Isolation

Human monocytes were isolated by using plastic adherence technique.Briefly, heparinized whole blood was freshly drawn and was diluted 1:1with phosphate-buffered saline (PBS). Blood/PBS was layered onFicoll-Hypaque and was then centrifuged at 400 g. The mononuclear celllayer was collected and washed with PBS then resuspended with in bovinecalf serum (BCS) in order to remove all the platelets. The mononuclearcells were then resuspended in 10% BCS/Dulbecco's Modified Eagle Medium(DMEM) to be plated on BCS-coated flask overnight to allow cells toadhere. After monocyte adherence, lymphocytes were removed by washingwith DMEM. Monocytes were then collected with 5 mM EDTA and counted.Before using, monocytes were resuspended in 10% BCS/DMEM and is thenready to be used.

RT-PCR

RNA was isolated using TRIzol reagent following to manufacturer'sinstructions. RT-PCR analysis was performed in a total volume of 25 uL.200 nM of forward and reverse primers and 50 ng/uL cDNA was used.Primers used were the same as Mansouri et al.³, which were: for iNOS,5′-ACATTGATGAGAAGCTGTCCCAC-3′ (sense) (SEQ ID NO: 1) and5′-CAAAGGCTGTGAGTCCTGCAC-3′ (anti-sense) (SEQ ID NO: 2); GAPDH,5′-CAGAACATCATCCCTGCCTCT-3′ (sense) (SEQ ID NO: 3) and5′-GCTTGACAAAGTGGTCGTTGAG-3′ (anti-sense) (SEQ ID NO: 4).

Example 4

We found that hyper-inflammatory macrophages exhibit remarkablecytotoxicity against B16 melanoma cells in vitro. Preliminaryobservations demonstrate decreased established/confluent B16 cells twodays after the introduction of hyper-inflammatory macrophages.

We postulated that these super-inflammatory macrophages possess theability to overcome and exploit the known interactions betweenmacrophages and melanoma and thus may be effective in the destruction ofneoplastic cells. We tested this hypothesis by co-culturing B16 melanomacells with thioglycollate-elicited intra-peritoneal macrophages fromwild type C57BL/6 and IL-6 knockout mice. A 4:1 E:T ratio was used perwell and the co-culture was carried out for forty-eight hours. In vivoelicited peritoneal macrophages were treated with a PPAR-γ agonist. FIG.23 illustrates graphs showing (A) iNOS expression and (B) TNF-αexpression of peritoneal macrophages isolated from C57BL/6 and IL6^(−/−)mice treated in vivo with a PPARγ agonist as indicated. Most remarkablewas the significant reduction in B16 cells despite being allowed to forma well-established mono-layer for twenty four hours (FIG. 24B, C) priorto the addition of hyper-inflammatory macrophages. There was nonoticeable reduction in the B16 melanoma cells when co-cultured withmacrophages obtained from wild-type mice (FIG. 24A, D).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. All patents, publications andreferences cited in the foregoing specification are herein incorporatedby reference in their entirety.

Having described the invention, the following is claimed:
 1. A method oftreating cancer or a tumor in a subject comprising: administering anamount of a PPARγ agonist and an IL-6/STAT3 signaling pathway antagonistto the subject effective to substantially inhibit STAT3 activation inthe macrophages of the subject; and administering an amount of aninflammatory or damaging insult to the cancer or tumor of the subjecteffective to induce hyper iNOS expression of the macrophages that are inor about the periphery of the cancer or tumor.
 2. The method of claim 1,the insult being administered directly to or about the periphery of thecancer or tumor.
 3. The method of claim 1, the insult comprising atleast one of trauma, physical stress, chemical stress, biologicalstress, or radiation.
 4. The method of claim 1, the PPARγ agonistcomprising a thiazolidinedione or a derivative thereof.
 5. The method ofclaim 1, the PPARγ agonist comprising at least one compound or apharmaceutically salt thereof selected from the group consisting of:(+)-5[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4thiazolidinedione;5-[4-[2-(5-ethylpyridin-2-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;(ciglitazone); 4-(2-naphthylmethyl)-1,2,3,5-oxathiadiazole-2-oxide;5-[4-[2-[(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]-5-methlthiazolidine-2,4-dione;5-[4-[2-[2,4dioxo-5-phenylthiazolidine-3-yl)ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-[(N-methyl-N-(phenoxycarbonyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-phenoxyethoxy)benzyl]thiazolidine-2,4-dione;5-[4-[2-(4-chorophenyl)ethylsulfonyl]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[[4-(3-hydroxy-1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl]thiazolidine-2,4-dione;5-[[2-(2-naphthylmethyl)benzoxazol]-5-ylmethyl]thiazolidine-2,4-dione;5-[4-[2-(3-phenylureido)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[2-(N-benzoxazol-2-yl)-N-metholamino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[2-(5-methyl-2-phenyloxazol-4-ylmethyl)benzofuran-5-ylmethyl]oxazolidine-2,4-dione;5-[4-[2-(N-methyl-N-(2-pyridyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;and5-[4-[2-(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]oxazolidine-2,4-dione.6. The method of claim 1, wherein the IL-6/STAT3 signaling pathwayantagonist comprises at least one of an antibody, peptide, or smallmolecule.
 7. The method of claim 1, wherein the IL-6/STAT3 signalingpathway antagonist is an HMG CoA reductase inhibitor.
 8. The method ofclaim 1, wherein the IL-6/STAT3 signaling pathway antagonist is a STAT3inhibitor.
 9. The method of claim 1, the cancer or tumor comprises skincancer.
 10. The method of claim 1, the PPARγ agonist being administeredto the skin cancer in a topical formulation.
 11. A method of treating aninfection in a subject comprising: administering an amount of a PPARγagonist and an IL-6/STAT3 signaling pathway antagonist to the subjecteffective to substantially increase hyper iNOS expressing cells at thesite of infection.
 12. The method of claim 11, the PPARγ agonistcomprising a thiazolidinedione or a derivative thereof.
 13. The methodof claim 11, the PPARγ agonist comprising at least one compound or apharmaceutically salt thereof selected from the group consisting of:(+)-5[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4thiazolidinedione;5-[4-[2-(5-ethylpyridin-2-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;(ciglitazone); 4-(2-naphthylmethyl)-1,2,3,5-oxathiadiazole-2-oxide;5-[4-[2-[(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]-5-methylthiazolidine-2,4-dione;5-[4-[2-[2,4dioxo-5-phenylthiazolidine-3-yl)ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-[(N-methyl-N-(phenoxycarbonyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-phenoxyethoxy)benzyl]thiazolidine-2,4-dione;5-[4-[2-(4-chorophenyl)ethylsulfonyl]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dine;5-[[4-(3-hydroxy-1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl]thiazolidine-2,4-dione;5-[[2-(2-naphthylmethyl)benzoxazol]-5-ylmethyl]thiazolidine-2,4-dione;5-[4-[2-(3-phenylureido)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[2-(N-benzoxazol-2-yl)-N-metholamino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[2-(5-methyl-2-phenyloxazol-4-ylmethyl)benzofuran-5-ylmethyl]oxazolidine-2,4-dione;5-[4-[2-(N-methyl-N-(2-pyridyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;and5-[4-[2-(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]oxazolidine-2,4-dione.14. The method of claim 11, wherein the IL-6/STAT3 signaling pathwayantagonist comprises at least one of an antibody, peptide, or smallmolecule.
 15. The method of claim 14, wherein the IL-6/STAT3 signalingpathway antagonist is an HMG CoA reductase inhibitor.
 16. The method ofclaim 14, wherein the IL-6/STAT3 signaling pathway antagonist is a STAT3inhibitor.
 17. The method of claim 11, the PPARγ agonist beingadministered to the site of infection in a topical formulation.
 18. Amethod for inhibiting unwanted wound repair in a subject comprisingadministering an amount of a PPARγ agonist and an IL-6/STAT3 signalingpathway antagonist to the subject effective to substantially inhibitSTAT3 activation in macrophages in or about the periphery of thesubject's wound.
 19. The method of claim 18, the PPARγ agonistcomprising a thiazolidinedione or a derivative thereof.
 20. The methodof claim 18, the PPARγ agonist comprising at least one compound or apharmaceutically salt thereof selected from the group consisting of:(+)-5[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4thiazolidinedione;5-[4-[2-(5-ethylpyridin-2-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[(1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;(ciglitazone); 4-(2-naphthylmethyl)-1,2,3,5-oxathiadiazole-2-oxide;5-[4-[2-[(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]-5-methylthiazolidine-2,4-dione;5-[4-[2-[2,4dioxo-5-phenylthiazolidine-3-yl)ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-[(N-methyl-N-(phenoxycarbonyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-phenoxyethoxy)benzyl]thiazolidine-2,4-dione;5-[4-[2-(4-chorophenyl)ethylsulfonyl]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[[4-(3-hydroxy-1-methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione;5-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl]thiazolidine-2,4-dione;5-[[2-(2-naphthylmethyl)benzoxazol]-5-ylmethyl]thiazolidine-2,4-dione;5-[4-[2-(3-phenylureido)ethoxyl]benzyl]thiazolidine-2,4-dione;5-[4-[2-(N-benzoxazol-2-yl)-N-metholamino]ethoxy]benzyl]thiazolidine-2,4-dione;5-[4-[3-(5-methyl-2-phenyloxazol-4-yl)propionyl]benzyl]thiazolidine-2,4-dione;5-[2-(5-methyl-2-phenyloxazol-4-ylmethyl)benzofuran-5-ylmethyl]oxazolidine-2,4-dione;5-[4-[2-(N-methyl-N-(2-pyridyl)amino]ethoxy]benzyl]thiazolidine-2,4-dione;and5-[4-[2-(N-(benzoxazol-2-yl)-N-methylamino]ethoxy]benzyl]oxazolidine-2,4-dione.21. The method of claim 18, wherein the IL-6/STAT3 signaling pathwayantagonist comprises at least one of an antibody, peptide, or smallmolecule.
 22. The method of claim 21, wherein the IL-6/STAT3 signalingpathway antagonist is an HMG CoA reductase inhibitor.
 23. The method ofclaim 21, wherein the IL-6/STATS signaling pathway antagonist is a STATSinhibitor.